mcherry plus a 30-nt 3′ linker  (Addgene inc)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Otoferlin interacts with Loop1.3. (A, Upper) Representative SDS/PAGE showing results of immunoprecipitation of otoferlin from lysate of HEK293 cells cotransfected with YFP-otoferlin and mCherry-Loop1.3. Both otoferlin and Loop1.3 precipitate. (A, Lower) Representative SDS/PAGE showing results of immunoprecipitation of YFP from lysate of HEK293 cotransfected with YFP and mCherry-Loop1.3. YFP but not Loop1.3 precipitates. IP, immunoprecipitated pellet; total lysate, lysate before immunoprecipitation. (B) Quantitation of coimmunoprecipitation showing the fraction of each protein in the IP and supernatant (n = 3 biological replicates; error = SE). (C) Cartoon depicting smCoBRA. Titration of a fluorescently labeled ligand onto immobilized YFP-otoferlin results in colocalized fluorescent puncta. The resulting saturation curve is fitted to obtain a dissociation constant Kd. (D) Dose–response for YFP-otoferlin titrated with Loop1.3 at increasing concentrations (0.1–50 µM) of free calcium. Each data point represents the mean of three biological replicates (n = 3). Experimental data are fit with a Langmuir isotherm (solid lines). Inset depicts 0–10 µM for clarity. (E) Determined Mander’s colocalization coefficients M1 (black) and M2 (red) for a dose–response of Loop1.3 titrated onto YFP-otoferlin (0.1 µM calcium). Inset depicts 0–10 µM. (F) Dose–response for immobilized synaptotagmin I titrated with Loop1.3 in the presence of 0.1 or 30 μM calcium (n = 3). Colocalization between Loop1.3 and YFP-otoferlin is included for comparison. (G) Dose–response for immobilized synaptotagmin I titrated with the loop II–III region of Cav2.2 in in the presence of 0.1–50 μM calcium (n = 3). Inset depicts 0–10 µM for clarity. MCC, Mander's correlation coefficients.

Article Snippet: Similarly, mCherry-Loop1.3 was created by first subcloning mCherry plus a 30-nt 3′ linker, supplied by Addgene (plasmid 29722), into CKAR via HindIII and KpnI sites and then subsequently subcloning Loop1.3 amino acids 752–891 into the KpnI and XbaI sites via PCR.

Techniques: SDS Page, Immunoprecipitation, Quantitation Assay, Titration, Labeling, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: The pathogenic mutation L1010 reduces otoferlin–Loop1.3 interaction. (A) Titration curve of Loop1.3 with immobilized YFP-otoferlinL1010P in 50 μM free calcium. (B) Representative photobleaching time course for colocalized YFP-otoferlinL1010P-mCherry-Loop1.3 puncta. Green arrowheads represent individual bleaching steps. (C) Photobleaching distributions for YFP-otoferlinL1010P-mCherry-Loop1.3 puncta in EDTA and calcium (n =2,300 puncta). (D) Dose–response for immobilized YFP-C2DL1010P titrated with t-SNAREs in the presence of 0.1 or 30 μM calcium (n = 3). Colocalization between t-SNAREs and YFP-otoferlin is included for comparison. (E) Dose–response for immobilized YFP-C2DL1010P titrated with Loop1.3 in the presence of 0.1 or 30 μM calcium (n = 3). Colocalization between Loop1.3 and YFP-otoferlin is included for comparison. (F) CD spectrum for WT C2D and C2DL1010P.

Article Snippet: Similarly, mCherry-Loop1.3 was created by first subcloning mCherry plus a 30-nt 3′ linker, supplied by Addgene (plasmid 29722), into CKAR via HindIII and KpnI sites and then subsequently subcloning Loop1.3 amino acids 752–891 into the KpnI and XbaI sites via PCR.

Techniques: Mutagenesis, Titration, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Otoferlin binds multiple Loop1.3 molecules. (A) Representative single-molecule photobleaching traces for Loop1.3-mCherry bound to YFP-otoferlin in the presence of 100 µM EDTA (Upper) and 200 µM calcium (Lower). Green arrowheads denote photobleaching events. (B) Single-molecule photobleaching distributions for EDTA and calcium conditions (n = 2,300).

Article Snippet: Similarly, mCherry-Loop1.3 was created by first subcloning mCherry plus a 30-nt 3′ linker, supplied by Addgene (plasmid 29722), into CKAR via HindIII and KpnI sites and then subsequently subcloning Loop1.3 amino acids 752–891 into the KpnI and XbaI sites via PCR.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Otoferlin binds t-SNAREs. t-SNARE binding curve in the presence of increasing free calcium concentrations (0.1–50 µM). (A) Experimental data are fit with a Langmuir isotherm (solid lines).Each experimental data point represents the mean value of n = 3. Inset depicts 0–10 μM for clarity. (B) Mander’s coefficients M1 (black) and M2 (red) for YFP-otoferlin–t-SNARE colocalization. Inset depicts 0–10 μM for clarity. (C) Titration of t-SNARE with immobilized YFP in the presence of 0.1 or 30 µM free calcium. Each experimental data point represents the mean value of n = 3. (D) Representative single-molecule photobleaching traces for mCherry–t-SNARE bound to YFP-otoferlin in the presence of 100 µM ethylenediaminetetraacetic acid (EDTA) (Upper) and 200 µM calcium (Lower). Green arrowheads denote photobleaching events. a.u., arbitrary units. (E) Single-molecule photobleaching distributions for EDTA and calcium conditions (n = 2,300).

Article Snippet: Similarly, mCherry-Loop1.3 was created by first subcloning mCherry plus a 30-nt 3′ linker, supplied by Addgene (plasmid 29722), into CKAR via HindIII and KpnI sites and then subsequently subcloning Loop1.3 amino acids 752–891 into the KpnI and XbaI sites via PCR.

Techniques: Binding Assay, Titration

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels

doi: 10.1073/pnas.1703240114

Figure Lengend Snippet: Single-molecule photobleaching distribution for t-SNAREs–mCherry bound to YFP-otoferlinL1010P in the presence of 100 μM EDTA and 200 μM free calcium.

Article Snippet: Similarly, mCherry-Loop1.3 was created by first subcloning mCherry plus a 30-nt 3′ linker, supplied by Addgene (plasmid 29722), into CKAR via HindIII and KpnI sites and then subsequently subcloning Loop1.3 amino acids 752–891 into the KpnI and XbaI sites via PCR.

Techniques: